CDN1163, a SERCA activator, causes intracellular Ca²⁺ leak, mitochondrial hyperpolarization and cell cycle arrest in mouse neuronal N2A cells

Objective: Activity or expression of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is diminished in some disease states such as cardiac failure and diabetes mellitus. A newly developed activator of SERCA, CDN1163, reportedly rescued or alleviated pathological conditions attributed to dysfunctional SERCA. We examined whether CDN1163 could relieve mouse neuronal N2A cell growth inhibition caused by cyclopiazonic acid (CPA, SERCA inhibitor). We also examined how CDN1163 affected cytosolic Ca²⁺, mitochondrial Ca²⁺ and mitochondrial membrane potential. Methods: Cell viability was measured by MTT assay and trypan blue exclusion test. Cytosolic Ca²⁺, mitochondrial Ca²⁺ and mitochondrial membrane potential were measured using fura 2, Rhod-2 and JC-1, respectively, as fluorescent probes. Results: CDN1163 (10 μM) itself suppressed cell proliferation, and did not alleviate CPA's inhibitory effect (and vice versa). Cell cycle was arrested at the G1 phase after CDN1163 treatment. CDN1163 treatment caused a slow yet persistent cytosolic [Ca²⁺] elevation partly due to Ca²⁺ release from an internal store other than the CPA-sensitive endoplasmic reticulum (ER). Treatment with CDN1163 for 3 h raised mitochondrial Ca²⁺ level and such increase was suppressed by MCU-i4 (an inhibitor of mitochondria Ca²⁺ uniporter, MCU), suggesting Ca²⁺ entered the mitochondrial matrix through MCU. Treatment of cells with CDN1163 up to 2 days resulted in mitochondrial hyperpolarization. Conclusion: CDN1163 caused internal Ca²⁺ leak, cytosolic Ca²⁺ overload, mitochondrial Ca²⁺ elevation and hyperpolarization, cell cycle arrest and cell growth inhibition.